Marimelia Porcionatto

TrkA glycosylation regulates receptor localization and activity

The human nerve growth factor receptor (TrkA) contains four potential N-glycosylation sites that are highly conserved within the Trk family of neurotrophin receptors, and nine additional sites that are less well conserved. Using a microscale deglycosylation assay, we show here that both conserved and variable N-glycosylation sites are used during maturation of TrkA. Glycosylation at these sites serves two distinct functions. First, glycosylation is necessary to prevent ligand-independent activation of TrkA. Unglycosylated TrkA core protein is phosphorylated even in the absence of ligand stimulation and displays constitutive kinase activity as well as constitutive interaction with the signaling molecules Shc and PLC-gamma. Second, glycosylation is required to localize TrkA to the cell surface, where it can trigger the Ras/Raf/MAP kinase cascade. Using confocal microscopy, we show that unglycosylated active Trk receptors are trapped intracellularly. Furthermore, the unglycosylated active TrkA receptors are unable to activate kinases in the Ras-MAP kinase pathway, MEK and Erk. Consistent with these biochemical observations, unglycosylated TrkA core protein does not promote neuronal differentiation in Trk PC12 cells even at high levels of constitutive catalytic activity.

Polarized signaling endosomes coordinate BDNF-induced chemotaxis of cerebellar precursors

During development, neural precursors migrate in response to positional cues such as growth factor gradients. However, the mechanisms that enable precursors to sense and respond to such gradients are poorly understood. Here we show that cerebellar granule cell precursors (GCPs) migrate along a gradient of brain-derived neurotrophic factor (BDNF), and we demonstrate that vesicle trafficking is critical for this chemotactic process. Activation of TrkB, the BDNF receptor, stimulates GCPs to secrete BDNF, thereby amplifying the ambient gradient. The BDNF gradient stimulates endocytosis of TrkB and associated signaling molecules, causing asymmetric accumulation of signaling endosomes at the subcellular location where BDNF concentration is maximal. Thus, regulated BDNF exocytosis and TrkB endocytosis enable precursors to polarize and migrate in a directed fashion along a shallow BDNF gradient.

Heparan sulfates and heparins: similar compounds performing the same functions in vertebrates and invertebrates?

The distribution and structure of heparan sulfate and heparin are briefly reviewed. Heparan sulfate is a ubiquitous compound of animal cells whose structure has been maintained throughout evolution, showing an enormous variability regarding the relative amounts of its disaccharide units. Heparin, on the other hand, is present only in a few tissues and species of the animal kingdom and in the form of granules inside organelles in the cytoplasm of special cells. Thus, the distribution as well as the main structural features of the molecule, including its main disaccharide unit, have been maintained through evolution. These and other studies led to the proposal that heparan sulfate may be involved in the cell-cell recognition phenomena and control of cell growth, whereas heparin may be involved in defense mechanisms against bacteria and other foreign materials. All indications obtained thus far suggest that these molecules perform the same functions in vertebrates and invertebrates.

Mesenchymal Stem Cell Therapy Modulates the Inflammatory Response in Experimental Traumatic Brain Injury

Therapy with mesenchymal stem cells (MSCs) has showed to be promising due to its immunomodulatory function. Traumatic brain injury (TBI) triggers immune response and release of inflammatory mediators, mainly cytokines, by glial cells creating a hostile microenvironment for endogenous neural stem cells (NSCs). We investigated the effects of factors secreted by MSCs on NSC in vitro and analyzed cytokines expression in vitro in a TBI model. Our in vitro results show that MSC-secreted factors increase NSC proliferation and induce higher expression of GFAP, indicating a tendency toward differentiation into astrocytes. In vivo experiments showed that MSC injection at an acute model of brain injury diminishes a broad profile of cytokines in the tissue, suggesting that MSC-secreted factors may modulate the inflammation at the injury site, which may be of interest to the development of a favorable microenvironment for endogenous NSC and consequently to repair the injured tissue.

Isolation and characterization of a heparin with high anticoagulant activity from Anomalocardia brasiliana

The isolation, some structural features, physicochemical properties and pharmacological activities of a heparin from Anomalocardia brasiliana are reported. It is shown that the mollusc heparin is very similar to those present in mammalian tissues with regard to chemical composition, physicochemical properties, pharmacological activities and susceptibility to heparinase and heparitinase II from Flavobacterium heparinum, as well as to the types of products formed by the action of these enzymes. Three significant quantitative differences were observed for the mollusc heparin when compared with the ones from mammalian origin, namely, a higher degree of binding with antithrombin III (45%), higher molecular weight (27-43 kDa) and higher anticoagulant activity (320 I.U./mg). The possible biological role of heparin is discussed in view of the present findings.

Effect of brown spider venom on basement membrane structures.

Loxoscelism or necrotic arachnidism are terms used to describe lesions and reactions induced by bites (envenomation) from spiders of the genus Loxosceles. Envenomation has been reported to provoke dermonecrosis and haemorrhage at the bite site and haemolysis, disseminated intravascular coagulation and renal failure. The purpose of this work was to study the effect of the venom of the brown spider Loxosceles intermedia on basement membrane structures and on its major constituent molecules. Light microscopy observations showed that L. intermedia venom obtained through electric shock, which reproduces two major signals of Loxoscelism in the laboratory, exhibits activity toward basement membrane structures in mouse Engelbreth-Holm-Swarm (EHS) sarcoma. Basement degradation was seen by a reduced periodic acid-Schiff (PAS) and alcian blue staining as well as by a reduced immunostaining for laminin when compared to control experiments. Electron microscopy studies confirmed the above results, showing the action of the venom on EHS-basement membranes and demonstrating that these tissue structures are susceptible to the venom. Using purified components of the basement membrane, we determined through SDS-PAGE and agarose gel that the venom is not active toward laminin or type IV collagen, but is capable of cleaving entactin and endothelial heparan sulphate proteoglycan. In addition, when EHS tissue was incubated with venom we detected a release of laminin into the supernatant, corroborating the occurrence of some basement membrane disruption. The venom-degrading effect on entactin was blocked by 1,10-phenanthroline, but not by other protease inhibitors such as PMSF, NEM or pepstatin-A. By using light microscopy associated with PAS staining we were able to identify that 1,10-phenanthroline also inhibits EHS-basement membrane disruption evoked by venom, corroborating that a metalloprotease of venom is involved in these effects. Degradation of these extracellular matrix molecules and the observed susceptibility of the basement membrane could lead to loss of vessel and glomerular integrity, resulting in haemorrhage and renal problems after envenomation.

In vivo and in vitro cytotoxicity of brown spider venom for blood vessel endothelial cells.

The effect of brown spider (Loxosceles intermedia) venom on endothelial cells was investigated in vivo and in vitro. Morphological and ultrastructural observations by light microscopy and transmission electron microscopy showed that the venom acts in vivo upon vessel endothelial cells of rabbits that were intradermally injected, evoking vessel instability, cytoplasmic endothelial cell vacuolization, and blebs. Likewise, treatment of rabbit endothelial cells in culture with the venom led to loss of adhesion of the cells to the substrate. Endothelial cells in culture were metabolically radiolabeled with sodium [35S]-sulfate and the sulfated compounds (proteoglycans and sulfated proteins) from medium, cell surface, and extracellular matrix (ECM) were analyzed. Agarose gel electrophoresis and SDS-PAGE showed that the venom is active on the ECM and on cell surface proteoglycans, shedding these molecules into the culture medium. In addition, when purified heparan sulfate proteoglycan (HSPG) and purified laminin-entactin (LN/ET) complex were incubated with the venom we observed a partial degradation of the protein core of HSPG as well as the hydrolysis of entactin. The above results suggest that the L. intermedia venom has a deleterious effect on the endothelium of vessels both in vivo and in culture, removing important constituents such as HSPG and entactin that are involved in the adhesion of endothelial cells and of subendothelial ECM organization.

The extracellular matrix provides directional cues for neuronal migration during cerebellar development

Normal central nervous system development relies on accurate intrinsic cellular programs as well as on extrinsic informative cues provided by extracellular molecules. Migration of neuronal progenitors from defined proliferative zones to their final location is a key event during embryonic and postnatal development. Extracellular matrix components play important roles in these processes, and interactions between neurons and extracellular matrix are fundamental for the normal development of the central nervous system. Guidance cues are provided by extracellular factors that orient neuronal migration. During cerebellar development, the extracellular matrix molecules laminin and fibronectin give support to neuronal precursor migration, while other molecules such as reelin, tenascin, and netrin orient their migration. Reelin and tenascin are extracellular matrix components that attract or repel neuronal precursors and axons during development through interaction with membrane receptors, and netrin associates with laminin and heparan sulfate proteoglycans, and binds to the extracellular matrix receptor integrins present on the neuronal surface. Altogether, the dynamic changes in the composition and distribution of extracellular matrix components provide external cues that direct neurons leaving their birthplaces to reach their correct final location. Understanding the molecular mechanisms that orient neurons to reach precisely their final location during development is fundamental to understand how neuronal misplacement leads to neurological diseases and eventually to find ways to treat them.

Fast preparation of nano-hydroxyapatite/superhydrophilic reduced graphene oxide composites for bioactive applications

A method for the direct electrodeposition of globular nano-hydroxyapatite (nHAp) onto reduced graphene oxide (RGO) is presented and a model for the specific growth preference is discussed. Results show that the carboxyl (carboxylic acid)/carboxylate functional groups attached directly to the RGO after oxygen plasma treatment were essential to accelerate the OH- formation and the deposition of globular nHAp crystals. High resolution scanning electron microscopy, energy dispersive X-ray and X-ray diffraction showed that homogeneous, highly crystalline, stoichiometric nHAp crystals, with preferential growth in the (002) plane direction, were formed without any thermal treatment. The nHAp/RGO composites were shown to be an appropriate surface for mesenchymal stem cell adhesion with active formation of membrane projections.

Post-translational Modifications of α5β1 Integrin by Glycosaminoglycan Chains THE α5β1 INTEGRIN IS A FACULTATIVE PROTEOGLYCAN

Cell-fibronectin interactions, mediated through several different receptors, have been implicated in a wide variety of cellular properties. Among the cell surface receptors for fibronectin, integrins are the best characterized, particularly the prototype α5β1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified α5β1 integrin as the only sulfated integrin among β1 integrin heterodimers expressed by the human melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and chemical reduction. The covalent nature of α5β1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 m NaCl, 4 mMgCl2, 8 m urea, and 6 m guanidine HCl. Based on deglycosylation procedures as chemical β-elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the α5β1 heterodimer and α5 and β1 integrin subunits were proteoglycans. The importance of α5β1sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells.